The structural basis for lesion search by the bacterial 8-oxoguanine DNA glycosylase MutM.

摘要

Maintenance of genome integrity is critical for normal cellular functions in all forms of life. DNA glycosylases serve as the first line of defense against most single-nucleobase lesions by catalyzing excision of the damaged base. MutM is the bacterial DNA glycosylase that specifically targets the major oxidative damage, 8-oxoguanine (oxoG), which differs from its million-fold more abundant progenitor guanine by only two atoms. How MutM and other DNA glycosylases effectively search through the vast genome to locate the rare damage sites remains a fundamental needle-in-a-haystack search problem in biology.;To unveil the lesion search mechanisms of MutM, we sought to structurally characterize MutM at all major stages of its interactions with undamaged and lesion-containing DNA. Previous studies have established that all DNA glycosylases extrude the target nucleobase into an extrahelical state bound to the enzyme active site in order for catalysis to proceed. The final lesion recognition state (LRC) of many DNA glycosylases, including that of MutM, has been elucidated structurally. In addition, intermolecular disulfide crosslinking has enabled the capture of a MutM interrogating an intrahelical undamaged nucleobase (the IC state). However, little is known at the atomic level about the initial encounter between MutM and an intrahelical lesion, the discrimination mechanisms against an extruded normal nucleobase, and how MutM translocates along the genome to find damaged DNA.;In this thesis, we report the crystal structures of four additional fleeting MutM-DNA interaction states trapped by disulfide crosslinking, which shed light on the DNA translocation, base extrusion and lesion detection steps of the lesion search process. Specifically, the Encounter Complexes (ECs) represent the first structures of a DNA glycosylase observed at the stage of interrogating an intrahelical, fully base-paired lesion. Sequence-matched sets of the EC, IC and LRC structures allow rigorous analysis of the conformational changes induced by an intrahelical oxoG upon binding to MutM, which underlie the kinetic preference of MutM for extruding a lesion versus an undamaged base. The Neighboring Complexes, in which an oxoG is placed in the neighborhood of the MutM target site, further reveal additional opportunities for MutM to detect intrahelical lesions embedded in duplex DNA. The Slanted Complexes illustrate an alternative intrahelical interaction state between MutM and normal or lesion-containing DNA, which raise a provocative model of strandwise DNA translocation that may facilitate lesion search. Finally, the Extrahelical G Complex shows how the MutM active site rejects an extrahelical undamaged G.;Taken together, the collection of MutM-DNA structures provides the first rather comprehensive glimpse of a DNA glycosylase interacting with both damage sites and nonspecific DNA substrates, thereby revealing the multi-layered mechanisms that ensure the high efficiency and precision of the lesion search process by MutM.